Taxanes are alkaloids possessing a taxane nucleus. The taxane nucleus comprises the three ring structure shown below which is also identified as 4,8,12,15,15-pentamethyl-tricyclo[9.3.1.0.sup.3,8 ]pentadecane. ##STR1##
At least 56 different taxanes have been identified in the literature. For example, in 1980, R. W. Miller authored an article which surveyed known taxus alkaloids and other taxane derivatives and reported for these compounds formulas, HNMR, MS, and x-ray data. The article by R. W. Miller, appearing at J. Nat. Prod. 43(4): 425-437 (1980) is incorporated herein by reference. The following publications which are also incorporated herein by reference identify and describe taxanes. M. G. Begley, E. A. Freeknall, and G. Pattenden, Acta Crystallogy, 40; 1745 (1984); D. P. Della Casa de Marcano and T. G. Halsall, Chem. Comm. 1382 (1970); D. G. I. Kingston, D. A. Hawkins, and L. Ovington, J. Nat. Prod. 45; 466 (1982); F. Gueritte-Voeglein, D. Guenard, and P. Potter. J. Nat. Prod. 50:(1):9-11 (1987); B. Lythgoe in "The Alkaloids" Ed. by R. H. F. Manske, Vol. 10, Academic Press, New York 1968, pp. 597-626; D. P. Della Casa de Marcano and T. G. Halsall, Chem. Commun. 1381 (1970); V. Senilh, S. Blechert, M. Colin, D. Guenard, F. Picot, P. Potier, and P. Varenne, J. Nat. Prod. 47(1) pp. 131-137 (1984); J. L. McLaughlin, R. W. Miller, R. G. Powell and C. R. Smith, Jr., J. Nat. Prod. 44:312-19 (1981); D. P. Della Casa de Marcano and T. G. Halsall, Chem. Comm. 365 (1975); C. H. Oliver Huang, David G. I. Kingston, Neal F. Magri, G. Samaranayake and F. E. Boetner, J. Nat. Prod. 49(4): 665-669 (1986).
The taxane series of molecules possess potent antitumor activity. Generally, the taxanes which have been studied for their antitumor activity have found use in the treatment of ovarian cancer and leukemia (W. P. MacGuire et al., Annals of Internal Medicine, vol. 111, pg. 273 (1989)). Taxanes are believed to exert their antitumor activity by inducing tubulin polymerization and forming extremely stable and nonfunctional microtubules which has an antiproliferative effect on taxane sensitive cells. (Eric K. Rowinsky et el., Journal of the National Cancer Institute, Vol. 82, No. 15, pp. 1247-1259 (1990)). Among the taxane molecules which have been studied most with respect to their antitumor activity are taxol, cephalomannine, desacetylcephalomannine, baccatin III, 10-desacetyl baccatin III and 10-desacetyltaxol. The structures of these taxanes are shown below: ##STR2##
The taxane compound known as taxol, was first reported to be isolated from the stem bark of the western yew Taxus brevifolia, a slow growing conifer. Its structure was elucidated by M. C. Wani et al., Journal of the American Chemical Society, Vol. 93, pp. 2325-2327, (1971).
Taxanes are commonly isolated from the bark of T. brevifolia collected in the wild. Because the concentration of specific taxanes in T. brevifolia is extremely low (for example, taxol is present in a concentration of between about 0.004% to about 0.02% based upon the dry weight of bark), large quantities of trees must be harvested and processed to produce even modest amounts of taxanes needed for research purposes. Furthermore, wild trees grow under very different conditions resulting in highly variable levels of taxanes produced in the bark. In addition, wild populations of trees are an unreliable source for taxanes because they are plagued with many uncertainties and risks such as forest fires, annual climatic variations, natural variations in taxol content in the different chemotypes of wild populations. Increased criticism from environmentalists concerning harvesting of wild plants also threatens the availability of T. brevifolia as an adequate source of taxanes. T. brevifolia, therefore, represents a nonrenewable and inconsistent source of taxanes.
Even more critical is the fact that extensive harvesting of wild trees risks the destruction of the germplasm essential for the future cultivation of T. brevifolia. Such harvesting could result in the loss of wild genes coding for proteins providing for such characteristics as disease and pest resistance, cold hardiness, high growth rates and tolerance to full sunlight and the extremes of drought and flooding and high taxol/taxane content. The preservation of these wild genes will be critical to long-term development of cost-effective taxol and other taxane production whether produced from cultivated plants, tissue culture or genetically modified microorganisms. Because of the critical role wild germplasm will serve in future production strategies, the preservation of wild populations should be considered an essential component of the development strategy for taxol and other taxane production.
Since the harvesting of wild populations of T. brevifolia yields such a limited supply of taxol, clinical experiments of taxol have been restricted to only a few specific chemotherapeutic applications. Lack of a stable and reliable source of taxol at a predictable cost will also significantly impede clinical utilization of the agent. Development of a sustainable, economic and reliable source for taxanes is imperative.
The potential of taxol as a cancer chemotherapeutic agent and the structural complexity of the taxol molecule has prompted a large effort directed toward its de novo synthesis. However, the molecular complexity of taxol suggests that a total synthesis of taxol from readily available raw material is not likely to be economically feasible.
A synthesis of the taxane ring skeleton is reported by R. A. Holton et al., at Journal of the American Chemical Society, 106 5731 (1984), and ibid, Vol. 110, pp. 6558-6560 (1988). However, these syntheses are deficient in that the final product lacks sufficient pharmacological activity to serve as an effective antitumor agent.
Semi-synthesis of taxol using 10-desacetyl baccatin III, a more abundant precursor isolated from the leaves of T. baccata (1 g/Kg fresh leaves), has been reported by French workers. Additionally, V. Senilh et al., C.R. Seances Acad. Sci. Ser. 2, Vol. 299, pp. 1039-1043 (1984); F. Gueritte-Voegelin, Tetrahedron, Vol. 42, pp. 4451-4460 (1986); Colin et al. European patent application 0 253 278 and Colin et al. European patent application 0 253 739 refer to the semisynthesis of taxol from 10-desacetyl baccatin III. These methods use taxane derivatives as the starting materials, and, therefore, suffer from the disadvantage that the approach requires isolation and purification of taxanes from a plant source followed by conversion of the purified taxane to taxol; this multistage preparation of taxol is more expensive than isolation of taxol directly from the plant material.
There have been efforts to develop new techniques for isolating taxanes such as taxol from plant matter. However, none of the methods reported to date provide for the adequate extraction of taxanes from a renewable source. For example, M. C. Wani et al., Journal of the American Chemical Society, Vol. 93, pp. 2325-2327 (1971), refers to the purification of taxanes from stem bark of Taxus brevifolia using normal phase column chromatography protocols. Normal phase column chromatography entails the use of a polar column packing to effect molecular separation.
National Cancer Institute Natural Products Branch paper NSC #125973 dated Jul. 15, 1983 also refers to the purification of taxol from bark, wood without bark, branches, twigs, needles, seeds/fruits and roots also from Taxus brevifolia.
A disadvantage of the method for isolating taxol described in the NSC #125973 paper and other publications is the reliance on the use of methylene chloride in the extraction process. Methylene chloride and other chlorinated hydrocarbons, such as chloroform, are recognized to be toxic and potentially carcinogenic. It is, therefore, desirable to avoid utilizing these solvents in the extraction and purification of taxanes from plant matter, both from the standpoint of exposure of these chemicals to personnel who carry out these procedures and from the standpoint of the potential exposure of patients to these chemicals via trace amounts not removed from the purified taxanes in the final dosage form of the taxane medications.
V. Senilh et al, Journal of Natural Products, Vol. 46, No. 1, pp. 131-137 (1984) refers to the purification of taxol and cephalomannine from the trunk of Taxus baccata L. The purification method which is reported by Senilh et al. is deficient in that further purification of specific taxanes using HPLC or crystallization is required even after multiple elutions of taxanes on a variety of normal phase chromatography packings has been performed.
There have been attempts to isolate taxanes from plant matter using reverse phase column chromatography protocols. See, e.g., Keith M. Witherup et al., Journal of Natural Products, Vol. 53, No. 5, pp. 1249-1253 (1990); Keith M. Witherup et al., Journal of Liquid Chromatography, Vol. 12, No. 11, pp. 2117-2132 (1989). These attempts are undesirable in that the taxanes are isolated in relatively low yield, approximately 50% based upon the theoretical yield of taxanes.
Reverse phase column chromatography entails the use of a nonpolar column packing to effect molecular separation. Another disadvantage of reverse phase chromatography is the difficulty in separating taxanes from the aqueous elution medium. The evaporation of an aqueous medium is both expensive and time consuming. Furthermore, the use of an aqueous medium hydrolyzes labile bonds, thereby lowering the yield of taxanes. Also, the aqueous medium used in the reverse phase chromatography step partially epimerizes the C-7 stereocenter. The epimers so produced are undesirable in that they have diminished pharmacological properties. Furthermore, these undesirable epimers are separated from pharmacologically useful taxanes only with additional expense and difficulty.
Separation is further complicated because desired taxane products coelute during the reverse phase chromatography step. Additional chromatography steps must therefore be performed to completely separate the taxanes, resulting in increased expense and effort.
Another shortcoming of known methods for isolating taxanes from plant matter is that the methods require that the plant matter be in substantially desiccated form. Present methods of drying plant matter promote the degradation of taxanes contained within the plant matter. Drying plant matter, therefore, contributes to decreased taxane yields.